This invention relates to an improved analytical method which effectuates the relatively rapid solubilization of hair and direct analysis of organic analytes, e.g., drugs of abuse, present in hair and other keratinized structures, e.g., fingernails and toenails, without effecting the structure of the analyte or being detrimental to biological analyte probes, e.g., antibody, RNA/DNA and bio-receptor probes. The analyte can be analyzed by adding the analyte probe directly to the solubilized keratin structure containing the analyte to determine the identity of the analyte as well as the extent and duration of its consumption by a subject.
In the past, hair analysis techniques for the detection of trace metals were developed that purported to provide information on an individual""s nutritional status. One objection to the use of these techniques is the difficulty of distinguishing between trace metals deposited in hair from the bloodstream and metals which have become embedded in hair through external contact with, for example, water and cosmetic agents. Consequently, these techniques are not considered useful by the medical community for diagnosing nutritional problems, and therefore have not been considered sufficiently accurate to determine the level of a particular trace metal consumed by a subject.
The problems with previous hair analysis techniques have caused reliance on urine and blood analysis techniques for the detection of ingested chemicals, e.g., drugs-of-abuse, medications and toxic chemicals, in a subject. However, these techniques also are known to be disadvantageous in that the duration and intensity of use or exposure cannot be ascertained. Urine and blood analysis, at best, can provide short term information concerning ingested drugs or chemicals such as drugs-of-abuse. In addition, there are also problems with the interpretation of such results. For example, the detection of a low level of ingested chemical in the urine could mean that a subject ingested a small amount of the drug or chemical very recently or a larger amount several days earlier. Thus, chronic drug use cannot be determined with these methods without repeated testing.
In response to the problems of establishing a reliable and accurate method that would measure both the duration and intensity of use of drugs-of-abuse, medications, toxic chemicals, etc., work performed by Dr. Werner A. Baumgartner, as reported in xe2x80x9cRadioimmunoassay of Hair for Determining Opiate Abuse Historiesxe2x80x9d, J. Nucl Med 20:749-752 (1979), determined that long-term histories of exposure to drugs-of-abuse can be obtained through the analysis of mammalian body hair, since these substances are xe2x80x9ctrappedxe2x80x9d within individual hair fibers during their synthesis. In this respect, hair was shown to act like a tape recorder, i.e., past exposure histories can be evaluated through sectional analysis of hair samples. It was found that heroin, once in the bloodstream, will find its way into hair as it is synthesized.
Thus, it was discovered in this study and confirmed by subsequent studies that a variety of chemicals, such as drugs-of-abuse, medications, toxic chemicals, etc., hereinafter collectively referred to as xe2x80x9canalytexe2x80x9d, are trapped by hair during its synthesis and that these substances are xe2x80x9clocked upxe2x80x9d in hair for essentially the duration of the hair. This was found to be true for head and body hair as well as for other keratinized structures such as fingernails. Suzuki et al., Forensic Sci. International, 24:9-16, 1984. These entrapped substances cannot be washed out of hair, and are completely released only upon the complete, or nearly complete, destruction of the hair fiber.
Prior art methods of extracting an analyte from hair included subjecting the hair to hot methanol solutions (Baumgartner et al., J. Nucl Med 20, 748, 1979) and by overnight incubation of hair in an alkaline or acid medium. D. Valente, et al., Clinical Chemistry, 1952, Vol. 27, No. 11, 1981. Prior methods also include the use of a mortar and pestle to release the entrapped analyte in conjunction with a solvent.
However, solvent extraction procedures suffer from several problems in accurately determining the presence and amount of an ingested analyte. One of these problems is that the solvent extraction methods frequently remove only a small unknown and variable fraction of the total analyte present in the hair sample. Such methods also tend to be time consuming, and generally involve elevated temperatures which may damage the analyte. Another disadvantage is that different analytes require different solvents for extraction. For example, a hair sample containing morphine, phencyclidine (xe2x80x9cPCPxe2x80x9d), cocaine and marijuana has to be extracted sequentially with several different solvents, which is a very time consuming procedure, particularly since the frequently toxic solvents have to be evaporated in expensive fume hoods before analysis can proceed.
Other methods and studies pertaining to the degradation of hair and hair analysis include:
O. Suzuki, et. al., in a publication by Elsevier Scientific Publishers Ireland Ltd., discloses a method for detecting methamphetamine and amphetamine in nail clippings or hair in which the substance was first washed in a mixture of methanol and water and dissolved in sodium hydroxide, followed by analysis of the extracted drug.
A. W. Holmes, in Textile Research Journal, 706-712, August 1964, discloses the degradation of human hair by papain using sodium sulfite as enzyme activator.
Annette M. Baumgartner, et al., in the Journal of Nuclear Medicine, 20:748-752, 1979, discloses the extraction of morphine and heroin from hair by pulverizing hair with a mortar and pestle followed by treatment with methanol.
D. Valente, et al., in Clinical Chemistry, Vol. 27, No. 11, 1981, discloses Dr. Baumgartner""s technique of subjecting hair to a treatment of hot methanol to effectuate extraction of drugs of abuse as well as the author""s technique of extracting morphine in an acid or alkaline medium.
A. M. Baumgartner, et al., in Journal of Forensic Sciences, p. 576-81, July 1981, discloses the extraction of PCP with mortar and pestle followed by treatment with methanol. The extracted PCP was then analyzed with RIA.
Smith et al., in Journal of Forensic Sciences, Vol. 26, No. 3, July 1981, pp. 582-586, disclose the testing of hair for the presence of phenobarbital, in which a single head hair was washed, dried, cut in 2 mm lengths and added to 0.2 ml 0.1% SDS/saline solution, and a sample assayed by radioimmunoassay.
W. A. Baumgartner, Black. et al., in J. Nucl Med 23: 790-892, 1982, discloses the extraction of cocaine from hair samples by refluxing the hair samples in ethanol followed by RIA analysis.
Ishiyama, et al., in Journal of Forensic Sciences, Vol. 28, No. 2, April 1983, pp. 380-385, disclose a method whereby hair from methamphetamine addicts was dissolved using 1.5 N hydrochloric acid at a pH between 1 and 2, followed by analysis using a gas chromatograph and mass spectrometry.
K. Puschel, et al., in Forensic Science International, 21 (1983) 181-186, discloses the dissolving of hair samples by exposure to sodium hydroxide and heat followed by analysis for the presence of morphine by RIA.
O. Suzuki, et al., in Journal of Forensic Sciences, Vol. 29, No. 2, April 1984, pp. 611-617, discloses the detection of methamphetamine and amphetamine in a single human hair by gas chromatography and chemical ionization mass spectrometry. The hair sample was first dissolved in a sodium hydroxide solution to which was added N-methylbenzylamine.
N. J. Haley et al., in Clin. Chem. 31/10, 1598-1600 (1985), discloses the analysis of hair for nicotine and cotinine, in which washed hair samples were dissolved in a buffer solution containing gelatin, sodium chloride, Tris and EDTA, and adjusted to pH 7.4. Samples were then analyzed by radioimmunoassay.
Sramek, Baumgartner, et al., in A.M.J. Psychiatry 142:8, August 1985, discloses the analysis of hair samples of psychiatric patients with methanol extraction and radioimmunoassay.
Baumgartner, et al., in Clinical Nuclear Medicine, Vol. 10, 4, September 1985, discloses the benefits of extracting entrapped drugs of abuse from hair followed by RIA analysis.
Gill, et al., in Nature, Vol. 318, p. 577 (1985) discloses the use of an SDS/proteinase k/dithiothreital mixture to extract DNA from whole blood, whole semen, vaginal fluid, hair roots, bloodstains and semen stains. The article states that xe2x80x9cno DNA could be isolated from hair shaftsxe2x80x9d.
Smith et al., in J. Forensic Sci. 1986, 31(4), 1269-73, discloses the detection of cocaine in perspiration, menstrual blood stains and hair using RIA.
M. Margio, et al., in xe2x80x9cDetermination of Morphine and Other Opioids in the Hair of Heroin Addicts by HPLC and MS/MSxe2x80x9d at the International Conference, University of Verona, Jun. 25-26, 1986, discloses various methods to assay morphine from hair samples.
M. Marigo, et al., in the Journal of Analytical Toxicology, Vol. 10, July/August 1986, discloses a method for the quantitative determination of morphine contained in the hair of heroin addicts, by means of heat-acid hydrolysis, pre-column dansyl derivatization, straight phase liquid chromatography and fluorescence detection.
Smith, et al., in Journal of Forensic Sciences, Vol. 31, No. 4, October 1986, pp. 1269-1273, disclose a method for the analysis of hair for the presence of drugs whereby hair samples were first washed, cut into small segments, mechanically pulverized for six minutes, refluxed in ethanol and the samples analyzed using radioimmunoassay.
M. Michalodinitrakis, Med.Sci.Law (1987), Vol. 27, No. 1, discloses the detection of cocaine in rats from the analysis of hair samples, which were dissolved upon exposure to 1.5 N HCl, which brought the pH value to 1-2, following incubation with 0.01 N Hcl at 37xc2x0 C. for one hour.
Pelli, et al., in Biomedical and Environmental Mass Spectrometry, Vol. 14, 63-68 (1987) discloses a procedure for the identification of morphine in the hair of heroin addicts in which hair is treated with diethylether and hydrochloric acid followed by dissolution of the dried extract in methanol.
Higuchi et al., in Nature, Vol. 332, p. 543 (1988) disclose a method for dissolving hair at pH 8 by the action of dithiothreitol, proteinase K, and 2% sodium dodecylsulfate in order to extract DNA from the digest by a complex chemical extraction method.
Also noted are certain patents, e.g., U.S. Pat. Nos. 3,986,926, 3,966,551, 3,939,040 and 3,623,950, which pertain to depilatory agents for the tanning of hides, and disclose the use of certain enzymes, including papain, in the dehairing process.
However, these and other prior art methods have proven disadvantageous for the reasons noted above and/or because they degrade the analyte probes (e.g., antibodies) of biological analytical methods, thereby preventing the use of such highly sensitive analytical techniques.
Thus, there exists a need for an analyte detection method that can rapidly and completely solubilize a certain analyte from keratinized structures of the body such as hair, fingernails and toenails of a subject and which permits direct analysis of the identity of the analyte and the duration of use of the analyte in, or exposure to, a subject, without destroying the analyte of interest and/or an analyte probe of biological analytical methods.
It is an object of the invention to provide a drug and chemical detection method.
It is another object of the invention to provide a drug and chemical hair analysis method.
It is another object of the invention to provide a reliable method of digesting head and body hair and other keratinized structures of the body and directly analyzing the identity and amount of analyte contained therein, and, where applicable, of determining the duration and extent of exposure of the analyte in a subject.
It is yet another object of the invention to provide a hair analysis method that solubilizes an analyte from the inner core of hair without causing damage to the analyte.
It is yet another object of the invention to provide a reliable hair digestion and direct analyte detection method that effectively permits the use of highly accurate biological analytical methods such as radioimmunoassay.
It is yet another object of the invention to provide a reliable hair analysis method that may be performed in a lesser period of time than known hair analysis methods.
It is yet another object of the invention to provide a drug detection method effective for use in the drug testing industry standard five-drug screen for marijuana, cocaine, opiates, methamphetamine and phencyclidine.
These and other objects are achieved by the novel analysis method according to the invention, which comprises preparing a mixture containing dithiothreitol (DTT) or dithioerythritol (DTE), an enzyme suitable for the digestion of keratinized structures and a sample of a keratinized structure; permitting DTT or DTE to activate the keratinized structure and/or the enzyme; permitting the enzyme to at least substantially digest the sample of keratinized structure to form a keratin digest solution; deactivating the DTT or DTE; and subjecting a portion of the keratin digest solution to analysis to detect the identity and amount of the analyte, if present, in the keratinized structure sample.
The preferred keratinized structure is hair. The enzyme may be any enzyme that, together with DTT or DTE, digests hair, and preferably is a protease including papain, chymopapain, or proteinase K. In order to accelerate the process, metal ions preferably in the form of metal salts may be added to the digest solution to deactivate any remaining DTT or DTE in the mixture which, if left active, would cleave the disulfide bond of the antibody of a biological analytical method such as an immunoassay, e.g. radioimmunoassay.
In accordance with the present invention, a method is provided that permits the rapid and complete digestion of head or body hair or other keratinized structure of an individual who may previously have ingested one or more analytes, followed by the identification of the analyte by known analytical biological probes such as the rapid and highly sensitive immunoassays. The release of the analyte into a digest solution from the interior of hair is effectuated according to the invention without damaging the analyte trapped within the organic matrix of the hair fiber which is to be analyzed, and without harmful effect on a subsequently-used probe (e.g., antibody) of a biological analytical method. The invention also permits the detection of past use patterns in a subject over extended periods of time without performing repeated testing as is necessary in conventional testing methods which measure the content of the analyte in samples of blood or urine. It has been found that the amount of analyte entrapped in hair of the same individual is directly proportional to the amount of analyte ingested.
A sample of a keratinized structure, e.g., hair, is first collected from a subject who may have ingested a particular analyte. Preferably, the hair sample is first washed by known methods to remove analyte or other drug or chemical which may have been deposited on the surface of the hair by external contact rather than by actual consumption. The hair sample is then subjected to treatment with a particular enzyme, together with a particular enzyme/substrate activator, so as to effectuate the complete or nearly complete digestion of the organic matrix of the hair fiber, known as keratin. The subject analyte that has been xe2x80x9centrappedxe2x80x9d within the organic matrix of the hair is then released into solution, or even if protein bound, the analyte is accessible to the antibody employed in protein-based analytical methods. In order to fully and accurately carry out the method according to the invention, a complete digestion of the sample is desirable.
Any enzyme that acts quickly to digest hair in conjunction with DTT or DTE without having a detrimental effect on the analyte is useful in the invention. In this regard, proteases are preferred for use in the invention. Most active, and therefore most preferred for use in the invention, are the proteases papain, chymopapain and proteinase K.
A number of other proteases have been found to be effective in the method according to the invention at low pH values (e.g., pH 7-9), namely, protease Type IV (bacterial, from Streptomyces caespitosus), Type VIII (from Bacillus subtilis), Type XI (proteinase K, fungal, from Tritirachium album), Type XIV (pronase, from Streptomyces griseus), Type XVI (from Bacillus subtilis), Type XVIII (Newlase, from Rhizopus species), Type XIX (from Aspergillus sojae), Type XXI (from Streptomyces griseus), Type XXIV (bacterial), Type XXVII (Nagarase), Type III (Prolase) and Type XXIII (from Aspergillus Oryzae) (all available from Sigma Chemical Co., St. Louis, Mo.).
As noted above, certain art-recognized procedures provide for the use of papain for use as a hair depilatory. These depilatory methods remove hair from hides and skin by softening it sufficiently so as to permit its ready removal by scraping or other mechanical means, and utilize inexpensive and less effective sulfhydryl enzyme and substrate activators such as thioglycolic acid or cysteine. These methods only partly degrade the hair and do not provide for the complete chemical digestion of the hair. A mere softening of the hair does not lead to the complete, or nearly complete, digestion of hair which is necessary in order to obtain a complete release of xe2x80x9centrappedxe2x80x9d analyte. Moreover, the sulfhydryl enzyme activators used in these depilatory methods are also harmful to certain biological analyte probes such as antibodies.
In contrast to these depilatory methods, the method of the present invention utilizes xe2x80x9cDTTxe2x80x9d (2,3 dihydroxybutane-1,4-dithiol) or its isomer xe2x80x9cDTExe2x80x9d (2,3 dihydroxybutane-1,4-dithiol) as the substrate and enzyme activating agent. Surprisingly, it has been found that the use of DTT or DTE in the process of the invention significantly enhances the digestion of the sample within a relatively short period of time, e.g., about three hours, resulting in the release of the analyte into the digest solution. Particularly surprising is that the invention may be used in the industry standard five-drug screen, in that it does not negatively impact upon any of those five drugs or their corresponding antibodies in the analysis step of the invention.
This high activity of the enzyme is believed to be due, at least in part, to the activation of the keratinized structure substrate itself by DTT and DTE, presumably by the action of DTT and DTE in opening up disulfide bonds in the keratinized structure, which facilitates enzymatic attack.
Once the protein of the keratinized structure has been completely or at least substantially digested, thereby releasing the analyte into the solution mixture, it has been found to be necessary to deactivate DTT/DTE and the sulfhydryl enzymes prior to subjecting the analyte to biological analytical probes, since the sulf-hydryl enzymes and enzyme/substrate activator(s) may interfere with the structural integrity of protein components of such methods.
The task of deactivating the sulfhydryl-dependent enzymes such as papain has proven difficult since after the digestion step, the enzymes are xe2x80x9cburiedxe2x80x9d in a xe2x80x9cseaxe2x80x9d of sulfhydryl groups belonging to the released hair proteins and enzyme/substrate activating agents. Known sulfhydryl blocking agents are ineffective in deactivating the enzymes, since the known sulfhydryl blockers tend to bind to the degraded hair proteins and DTT or DTE and not necessarily to the enzyme sulfhydryl sites critical for blocking the activity of the enzymes. Thus, it is not possible to effectively utilize the protein-based analytical methods if the enzyme sulfhydryl sites are still active.
It was quite surprising, therefore, that DTT and DTE act not only to activate enzymes and/or the keratinized structure substrate causing unexpectedly high hair digestion activity, but that they also may act to deactivate the enzyme by a direct or indirect (enzyme self-deactivation) mechanism after the enzyme effectuates the complete, or nearly complete, digestion of the hair protein. Typically, the enzyme deactivation occurs within about four to five hours after exposure of the DTT or DTE to the enzyme, which is a sufficient amount of time for the enzyme to effectuate the digestion of the hair sample. Once the enzyme has been deactivated, it has been found that the enzyme cannot be reactivated or regenerated by exposure to fresh DTT or DTE.
Deactivation of at least certain of the non-sulfhydryl dependent proteases, e.g., proteinase K, by its inhibitor, phenylmethyl sulfonyl chloride, is generally not required since the enzyme has not been found to be active against the antibodies used in protein based immunoassay techniques.
It also has been found that active DTT or DTE present in the hair digest solution constitutes a hazard to the structure and activity of other proteins to which it is exposed, e.g., antibodies utilized in radioimmunoassay. Thus, it was a further surprising result that DTT or DTE in the reaction mixture may not only act to deactivate the enzyme, but itself deactivates in the digest solution without the introduction of an inhibitor. Typically, DTT and DTE will deactivate after the hair sample has been digested, less than about 14 hours after its first exposure to the enzyme depending on the various concentrations and amounts of the enzyme and DTT or DTE utilized, the pH, temperature, amount of hair sample, etc.
Thus, in accordance with the method of the invention, complete digestion can be carried out in a relatively short period of time, e.g., overnight, and the digest solution, which includes the released analyte of interest, can be directly subjected, effectively and accurately, to protein-based ligand assay analysis methods the next morning. Typically, the entire method, from the washing of hair samples to the identification of the analyte, should take no longer than about 16-20 hours. Little or no intervention by the individual performing the method is needed to release the analyte from the hair sample once the enzyme and DTT or DTE come into contact with the hair sample.
Alternatively, it has been discovered that the addition of certain metal ions, typically in the form of metal salts, to the digest solution results in a rapid deactivation of DTT or DTE. The addition of low amounts of such metal salts to the digest solution after digestion of the sample significantly accelerates the time in which the hair digest mixture can be subjected to the immunoassay method since it is not necessary to wait for DTT or DTE to deactivate on its own. This discovery is particularly surprising as not all metal ions are effective in deactivating DTT and DTE, or otherwise are not useful in the invention.
Most effective for use in the invention are certain metal salts which surprisingly do not precipitate out of the solution after chemically linking with, and deactivating, DTT/DTE. It is important that precipitation not occur in the digest solution because such precipitation could result in a loss of analyte by adsorption. Preferably, precipitation is prevented by maintaining the pH of the digest solution at about 6-8, and most preferably at about 7. One way this may be accomplished is by the addition of one molar Trizma base. Surprisingly, the most preferable pH of about 7 is also the optimum pH for the performance of radioimmunoassay (xe2x80x9cRIAxe2x80x9d).
In addition to Cu++ salts (e.g., copper sulfate) as described in Applicant""s U.S. Pat. Nos. 5,466,579 and 5,324,642, salts of Zn++ (e.g., zinc sulfate and zinc nitrate); Mn++ (e.g., manganese sulfate); Fe++ (e.g., ferric sulfate and ferric chloride); and Fe+++ (e.g., ferrous sulfate) are particularly effective and preferred for use in the invention. Also effective for use in the invention are salts of Pb++ (e.g., lead acetate and lead nitrate); Cd++ (e.g., cadmium chloride); Hg++ (e.g., mercuric chloride); Ag++ (e.g., silver nitrate); and Co++ (e.g., cobalt chloride).
Typically, about 100 microliters of metal salt (10 mg/ml) is added to 1 ml of hair digest solution about 4 to 5 hours after contacting the enzyme and DTT (or DTE) with the hair sample so as to permit the enzyme and DTT (or DTE) sufficient time to digest the hair sample.
Similarly, any salt of arsenite, and preferably sodium arsenite (NaAs02), may be utilized in the invention to remove residual DTT or DTE by formation of a precipitable compound. Typically, 100 microliters of a 100 mg/ml solution of sodium arsenite is added to 1 ml of hair digest solution to effectuate the deactivation of DTT and DTE. However, arsenite is not preferred because a precipitate usually develops. It may, however, be useful in certain circumstances.
Once the rapid and effective digestion of the sample occurs, the digest solution may then be subjected to direct analysis by art recognized protein-based analytical methods such as RIA. Such methods are preferred for use in the invention because RIA and related immuno- or ligand assays are currently the only known mass production procedures having the required sensitivity and convenience for measuring the low concentrations of analytes contained in hair samples. The use of these methods is preferred because only about 0.5 to 1.0 mg. of hair is necessary for analysis by RIA and other protein-based analytical methods.
Other analytical methods may be utilized in place of or in addition to the protein-based analytical methods, including instrumental means such as chromatography, mass spectrometry, etc. In particular, these methods may be used to confirm positive results obtained in RIA. Because these methods are not protein-based, the steps of deactivation of the enzyme and DTT or DTE is not necessary when using non-protein-based analytical techniques. However, the speed and gentleness of the extraction method according to the invention and the ability to quantitate the extraction efficiency through the inclusion of a xe2x80x9cspikexe2x80x9d, i.e., the inclusion of a known amount of deuterated analyte, makes the presently disclosed digestion method also the method of choice for instrumental analysis methods such as gas chromatography and mass spectrometry.
The method according to the invention has been found to be effective in detecting the use and prior use of drugs of abuse such as cocaine, morphine/heroin, marijuana, phencyclidine or xe2x80x9cPCPxe2x80x9d, methaqualone and methamphetamine. Moreover, the method according to the invention has been found to be effective in determining prior usage of prescription drugs such as digoxin, methadone and benzodiazepines. It is contemplated that any organic analyte present in the bloodstream of an individual which is transferred to the hair during its synthesis can be extracted and analyzed in accordance with the method of the invention.
In carrying out the method, it is preferred that an aqueous solution of about 110 mg DTT or DTE/10 ml water be used, although concentrations of DTT or DTE of about 50-200 mg/10 ml water are effective in the method. It is preferred that the weight ratio of DTT or DTE to papain or chymopapain be about 110:2 [when enzyme purity is 16-40 BAEE units/mg protein], although efficacious results have been observed at weight ratios of DTT or DTE to papain or chymopapain ranging between about 110:1 to about 110:4. With respect to proteinase K and other proteases, it is preferred that the weight ratio of DTT or DTE to proteinase K (or other proteases) be about 1200:1 (when enzyme purity is 10-20 units per mg. protein), although weight ratios of 1200:0.5 to about 1200:2 also will be effective.
The concentration of hair protein is preferably kept constant at about 10 mg hair/cc of digest solution so as to prevent variable matrix effects in a subsequently utilized protein-based analytical method.
The enzymatic digestion of hair and other keratinized structures, according to the method of the invention, may be conducted at low temperatures and near neutral pH. When papain, chymopapain or other sulfhydryl dependent enzyme is utilized as the enzyme, the method may be performed at a temperature of between about 20xc2x0 C. and 40xc2x0 C., and at a pH between about pH 8.8 and 10.5. Preferably, the pH of the method is between about 8.8 and 9.5 at a temperature of about 37xc2x0 C.
When proteinase K or other proteases are utilized as the enzyme, it is preferable to perform the method between about 20 and 40 degrees centigrade and at a pH between about 7 and 9. When the temperature is about 37 degrees centigrade and the pH about 7.0 or below, the risk of altering the structure of a particular analyte is at a minimum. Other enzymes which digest hair under neutral or acid conditions include: Protease Type XIV (Pronase), Type III (Prolase), Type IV, Type VIII, Type XVI, Type XVIII, Type XIX, Type XXIV, Type XXVII (Nagarse), Type XXVIII, Type XXI and Type XXIII.
Under certain circumstances, it is advantageous to perform the method according to the invention at a lower than usual pH in order to preserve the chemical structure of the analyte. As stated above, the digestion typically will occur at a pH between about 8.8 and 10.5 when a sulfhydryl dependent enzyme (e.g., papain) is utilized and between 7 and 9 when a protease such as Proteinase K is utilized. At any pH in either of these ranges, however, certain analytes may become unstable or hydrolyze to a different form, which may impact on the measure of both quantity and quality of the analyte in the subsequent analysis step.
Thus, for example, at a pH of about 7, the heroin metabolite, 6-monoacetylmorphine, breaks down rapidly to morphine, thereby making heroin users indistinguishable from morphine users. In addition, the stability of cocaine is also quite pH dependent, and the performance of the method at an improper pH may lead to a false interpretation of a positive cocaine result.
Ingested cocaine naturally hydrolyzes to benzoylecgonine in the blood and eventually ends up entrapped in hair both as cocaine and benzoylecgonine. The digestion of hair from a cocaine user will lead to the release of both cocaine and benzoylecgonine from the hair into solution thereby resulting in the conclusion that a positive cocaine hair analysis result was caused by cocaine ingestion.
In situations where an individual has not ingested cocaine but is only exposed to cocaine environmentally, contaminated hair will contain only cocaine and not the benzoylecgonine metabolite. Thus, the absence of benzoylecgonine confirms lack of drug use. Conversely, the presence of benzoylecgonine refutes any claim that a positive result was caused by the external contamination of a hair sample, i.e., by faulty, ineffective washing of the sample by the laboratory to remove cocaine contaminants deposited from the environment. However, cocaine tends to hydrolyze to benzoylecgonine at a pH above about 6.5 and a temperature of 37xc2x0 C. As a result, the certainty of distinguishing between drug use and external contamination can only be achieved if the pH of the digest solution is maintained so as to avoid the production of significant quantities of benzoylecgonine.
Thus, in the case of certain analytes such as cocaine which may be chemically altered by a higher pH, it is desirable to perform the method of the invention at a pH which avoids hydrolysis or other chemical reaction of externally deposited analyte which inadvertently has ended up in the digest solution. In the case of cocaine, performance of the method at a pH below about 6.6 at 37xc2x0 C. will ensure that the benzoylecgonine in the sample is directly related to ingested cocaine and not to externally deposited cocaine.
According to the invention it has been found that certain biological detergent compounds useful for solubilizing biological membrane components aid in the digestion of hair at a relatively low pH while not interfering with enzymatic activity or the antibody-antigen reaction which will influence the sensitivity of the immunoassay. This is surprising and unexpected since other biological detergents have been found to be unsuitable for use in the invention because they are ineffective in aiding digestion at the desired low pH, they deactivate proteinase K or other hair protein digestion enzymes and/or they impact on the binding of the analyte by the antibody thereby drastically reducing the sensitivity of the immunoassay. See, e.g., Higuchi, R. et al., xe2x80x9cDNA Typing From Single Hairsxe2x80x9d, Nature, 332:543-546, 1988.
These biological detergents, together with an appropriate enzyme (e.g., protease and sulfhydryl enzymes) and activator (e.g., DTT and DTE), are effective in aiding the digestion of the hair sample at a lowered pH in the range of about 5.8 and 8. Those detergents found to be useful in the invention include the bile acid detergents, such as glycocholic acid, cholic acid, taurocholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid and salts thereof, including sodium salts. Other detergents effective for use in the invention are sulfo-betaines, such as the Zwittergents(copyright), and betaines, such as Empigen BB (N-dodecyl-N,N dimethylglycine) (all available from Calbiochem Corp., La Jolla, Calif.).
Still other detergents which are useful in aiding the digestion of hair according to the invention at a relatively lower pH are the alkylglucosides, including hexyl-xcex2-D-glucopyranoside, heptyl-xcex2-D-glucopyranoside, octyl-xcex2-D-glucopyranoside, nonyl-xcex2-D-glucopyranoside, decyl-xcex2-D-glucopyranoside, dodecyl-xcex2-D-maltoside and octyl-xcex2-D-thioglucopyranoside (OSGP). Mixtures of alkylglucosides, such as the product ELUGENT (Calbiochem), are also effective.
Particularly preferred for use in the invention are the bile acids cholic acid and glycocholic acid which aid in the digestion of hair at a pH in the range of about 6.3-8. The deoxycholates such as deoxycholic acid and glycodeoxycholic acid are effective in aiding in the digestion of hair at a pH above about 7.
As discussed above, the hydrolysis of cocaine occurs at a pH of about 6.6 and above at 37xc2x0 C. Thus, to avoid significant hydrolysis of cocaine to benzoylecgonine, the digestion preferably is performed at a pH of 6.5 or below, and typically in the range of about 6.3-6.5.
Surprisingly, certain of these detergents are efficacious when the industry standard five-drug screen for the most common drugs of abuse in the United States, i.e., marijuana, cocaine, phencyclidine, methamphetamine and opiates, is performed using the method of the invention. Thus, they do not impact on any of the analytes or antibodies involved in the five-drug screen, and do not result in false negatives or positives. This is particularly surprising given the fact that the chemical nature of these five analytes ranges from highly lipidic drugs such as PCP and marijuana to highly water soluble drugs such as benzoylecgonine and morphine.
The particular detergents most effective for use in the five-drug screen are cholate, deoxycholate, cholic acid, deoxycholic acid, octyl-xcex2-D-glucopyranoside and octyl-xcex2-D-thioglucopyranoside. The bile acid detergents, alkylglucosides, sulfobetaines and betaines are most preferred when a screen is performed that includes cocaine, opiates, phencyclidine and methamphetamine. In a screen solely for cocaine, the preferred detergents are cholic acid, Zwittergents(copyright), alkylglucoides, and N-dodecyl-N,N dimethylglycine.
Of the sulfo-betaine detergents manufactured by Calbiochem Corp. of La Jolla, Calif., Zwittergent(copyright) SB3-14 (CAS Registry No. 14933-09-6, N-tetradecylsulfobetaine or 3-(dodecyldimethylammonio) propane-1-sulfonate.) is preferred. Digestion of hair using the Zwittergent(copyright) sulfo-betaine detergents typically occurs at about pH 6.3 at 37xc2x0 C. Zwittergents(copyright) are of the class of detergents known as sulfo-betaines having the general structure: 
wherein x may be any number which provides an effective biological detergent. Preferred are those compounds wherein x is in the range of 7-16. Most preferred is the detergent when x is 14. Other Zwittergent(copyright) sulfo-betaine detergents useful in the invention include:
1. Zwittergent(copyright) SB3-08 wherein x=7 [N-octyl sulfo betaine or 3-(Octyldimethylammonio) propane -1-sulfonate] [CAS # 15178-76-43].
2. Zwittergent(copyright) SB3-10 wherein x=9 [N-D-dodecylsulfobetaine or 3-(dodecyldimethylammonio) propane-1-sulfonate] [CAS Registry # 15163-36-7].
3. Zwittergent(copyright) SB3-12 wherein x=11 [N-dodecylsulfobetaine or 3-(Dodecyldimethylammonio) propane-1- sulfonate] [CAS #14933-09-6].
4. Zwittergent(copyright) SB3-14 wherein x=13 [N-tetradecylsulfobetaine or 3-(Dodecyldimethylammonio) propane-1-sulfonate] [CAS # 14933-09-6].
5. Zwittergent(copyright) SB3-16 wherein x=15 [N-Hexadecylsulfobetaine or 3-(Hexadecyldimethylammonio) propane-1-sulfonate] [CAS #2281-11-0]
The bile acid detergents and Calbiochem Zwittergents(copyright) SB3-8, SB-10, SB3-12, SB3-14 and SB3-16 (x in the range of 7-16) are preferred in effectuating the digestion of hair at a relatively lower pH which is desirable in methamphetamine, PCP and opiates assays. The cholate and Calbiochem Zwittergents(copyright) are preferred for use in the cocaine assay. The cholate and deoxycholate detergents are preferred for use in marijuana screening assays.
In practice, the biological detergent is mixed with the aqueous digest solution of the activator such as DTT (or DTE) and the enzyme (preferably proteinase K) prior to contact of the solution with the hair sample at a preferred temperature range of about 30-40xc2x0 C. as described herein. Typically, about 1-2 mg of biological detergent is added to about 1 cc of digest solution.
In another embodiment according to the invention, an ion exchange resin is employed to remove from the hair digest solution a substance which uniquely interferes with the marijuana assay. This interference has occurred with all currently available commercial RIA kits suitable for detecting cannabinoids. The interfering substance, effectively present in every hair sample in varying amounts from individual to individual and believed to be naturally occurring in hair, appears to interfere with the assay as a result of a cross reaction with the marijuana RIA antibody (i.e., specific to cannabinoids) rather than by matrix effects. This appears to be the case because dilution of the interfering substance produces an asymptotic curve which appears identical in shape to the calibration curve obtained. with the carboxytetrahydro-cannabinol (xe2x80x9ccarboxy-THCxe2x80x9d) standard used for an RIA marijuana assay, rather than producing an S-shaped dilution curve which would have been expected if matrix effects were the cause of the interference. It appears that this interfering substance is lipidic and bears a close resemblance in structure at the immunological binding site to carboxy-THC.
Because of its similarity to carboxy-THC, and other cannabinoids such as tetrahydrocannabinol (xe2x80x9cTHCxe2x80x9d), the interfering substance results in false positive results in assays on hair digests using RIA to determine marijuana exposure. In other words, the RIA erroneously will identify the interfering substance as a cannabinoid from exposure to marijuana, even in individuals not exposed to marijuana. Thus, it is necessary when performing an RIA assay for marijuana exposure to somehow remove the interfering substance from the digest solution prior to subjecting the solution to immunoassay analysis.
Since the presence of this interfering substance in hair is a new discovery, there is no method known to the art for removing it. Removal of the interfering substance from the digest solution is further complicated by its similarity to lipidic carboxy-THC and THC, the more common diagnostic analytes in a marijuana assay. Many filtering techniques with the capability of filtering out undesirable substances are not effective in removing the interfering substance from the digest solution, because they either do not effectively remove the interfering substance, and/or because they remove the analytes, e.g., THC and Carboxy-THC.
It was thus surprising that there exists in hair an analyte indicative of marijuana exposure which does not possess many of the same lipidic properties of the interfering cross reacting substance but yet will react with the cannabinoid antibody used in the RIA assay for marijuana. The exact chemical structure of this immunoreactive substance(s), or modified-lipidic marijuana analyte, is unknown. However, its presence in the hair sample will indicate marijuana use.
Also surprising is the discovery that the interfering substance may be removed from the digest solution without removing the newly discovered modified-lipidic marijuana analyte by the use of certain commercially available ion exchange resins. It has been found that suspensions of certain ion exchange resins upon contact with the digest solution will remove the interfering substance along with certain cannabinoids such as THC from the digest solution, but will leave in the digest solution other diagnostic cannabinoids, such as the modified lipidic marijuana analyte, which then may be detected by commercially available RIA kits utilizing a cannabinoid antibody. The use of these ion exchange resins to remove the interfering substance, thus permitting the detection of the modified-lipidic marijuana analytes in the RIA assay without interference, is both convenient and cost effective.
Effective ion exchange resins are both anionic and cationic. They generally are commercially available, such as from Sigma Chemical Co. of St. Louis, Mo. However, they have been found to be most effective not in the form in which they are commercially available (generally course, fast settling particles), or in the way they are generally used (e.g., packed columns) but when contacted with the digest solution in the form of a suspension, e.g., when broken apart into much smaller, slowly settling particles by vigorous stirring and made into a fine suspension of these small particles. Effective ion exchange resins include:
1. Anion exchangers on dextrose such as DEAE Sephadex(copyright) (A-25 and A-50 Diethylaminoethyl Sephadex(copyright)) and QAE Sephadex(copyright) (Q-50 Diethyl-[2-hydroxypropyl] aminoethyl Sephadex(copyright));
2. Anion exchangers on agarose such as DEAE Sepharose(copyright) CL-6B (Diethylaminoethyl Sepharose(copyright)) and Q Sepharose(copyright);
3. Anion exchangers on cellulose such as DEAE-Sephacel(copyright) (Diethylaminoethyl Sephacel(copyright)); Ecteola Cellulose (Epichlorohydrin Triethanolamine Cellulose); PEI Cellulose (Polyethyleneimine Cellulose); QAE Cellulose (Diethyl-[2-Hydroxypropyl]aminoethyl Cellulose); and DEAE cellulose;
4. Cation exchangers on dextran, such as SP Sephadex(copyright) C25 (Sulfopropyl Sephadex(copyright))
5. Strongly acidic cation exchangers on polystyrene, such as Amberlite(copyright) 200 (active group: sulfonic acid, sodium form); Dowex(copyright) HCR-S (active group: nuclear sulfonic acid, hydrogen form) and Dowex(copyright) macroporous resin (active group: nuclear sulfonic acid, hydrogen form).
6. Specialty Exchangers such as benzyl DEAE cellulose (Benzyl Diethylaminoethyl cellulose) and TEAE cellulose (triethylaminocellulose).
The concentration of the resin will vary depending on the resin employed. Concentrations may vary up to about 50% (wt./vol.). For example,.a suspension of DEAE Sephadex(copyright) A25 in deionized water preferably is prepared using about 3 to 9 gm. resin/100 ml water. The suspension is allowed to swell. At least one hour of swelling at room temperature has been found to be adequate. After swelling, the suspension is stirred or shaken vigorously for at least thirty minutes and preferably up to about sixty minutes until a very fine suspension is obtained.
Preferably, the suspension has a settling time in the range of 50-60 minutes compared to about 10-15 minutes of an aqueous suspension of an unshaken resin. Vigorous stirring or any other method which results in the break up of the resin into small particles provides the optimum results in removing the interfering substance from the digest solution.
To achieve removal of the interfering substance, approximately equal parts of hair digest solution and fine suspension are mixed together. The ratio of suspension to hair digest solution, however, will vary depending on the resin used and its concentration in the resin suspension. Generally, the ratio of digest solution to suspension is 4 to 3, if a suspension of 3 to 9% (wt./vol.) is used. The mixture is rotated so that the suspension and digest solution are in direct contact. Distilled water is then added, the mixture centrifuged and a sample of the supernatant from the resinated digest solution assayed according to the invention.
While the method according to the invention for removing the interfering substance has been described in connection with a digest solution, it is recognized that the ion exchange resin suspension may be used in any hair analysis method where removal of the interfering substance is desirable or necessary, provided that the analytes are ultimately contained in an aqueous albumin solution near neutral pH as required for the RIA assay. The resin suspensions may be used, for example, together with known hair extraction (acid, base or solvent) methods or any other method which disrupts a hair sample and results in the release into a solution at least a portion of the contents of a hair sample which includes the interfering substance.
In contrast to other available analyte detection methods such as urine and blood analysis, the method in accordance with the invention permits detection of exposure to an analyte over a period of time, and is therefore quite beneficial in detecting chronic drug use. Since hair is known to grow at a rate of about 0.3-0.4 mm/day or about 1.0-1.3 cm/month, it is possible to measure consumption or exposure as far back as the hair length permits by evaluating snippets of hair of various lengths, and the use of highly sensitive protein-based analytical methods permits analysis of small samples of analyte contained in the small snippets of hair.
Through sectional analysis, the method of the invention provides a relatively permanent record and evidence of a pattern of drug use, or the prior ingestion of other substances, for periods ranging from several days to months or even years after last use. The history of such exposure can be made as detailed as desired by analyzing suitably short sections of hair representing different periods of growth. In this way, prior usage over time, and the extent of such use, can be determined.
Although the use of head hair is preferred for use in the invention due to its length and accessibility, it is possible to utilize any other body hair or fingernails in the method of the invention. Thus, it is not practically possible to evade testing by the method of the invention by shaving one""s head or body hair.
However, treatments such as perming and dyeing may increase the rate of digestion of hair subjected to the method according to the invention. In some cases, some analyte may be lost prior to performing the procedure due to such treatments. When the subject hair has been so altered, an increase in digestion rate is evident and an appropriate correction factor may be applied based upon known rates of normal hair digestion.
Certain other cosmetic agents, such as certain relaxing agents, may cause hair to become resistant to digestion. Such resistance may be overcome in some instances by increasing the quantity of enzyme to be used. Preferably, proteinase K is utilized as the enzyme when such resistance to digestion is encountered.
Alternatively, when it is not possible to make use of body hair or in some instance when the use of hair is not desirable, the use of other keratinized tissue such as fingernails and toenails may be used in the invention. In this regard, the effective ratio of DTT or DTE to enzyme needed to digest fingernails and toenails in order to release the analyte is about the same as for use with hair. Once the fingernail or toenail samples are digested in accordance with the method described herein, the released analyte may be analyzed by a desired analytical method.
In another aspect of the invention, it has been surprisingly discovered that melanin granules contained in hair can be dissolved by the combined action of the enzyme (preferably papain), DTT or DTE and ethylene diamine tetraacetic acid (EDTA), the latter at a concentration of about 5 mg EDTA/ml of digest solution. Since certain analytes or drugs of abuse such as PCP have been discovered to accumulate in the granules contained in mouse hair, dissolution of the granules, which also are present in the digest solution of human hair, may possibly be effectuated and the analyte contained in the human hair granule identified.
In accordance with this aspect of the invention, a hair digest solution is obtained as described above, and the melanin granules recovered from the hair digest solution, e.g., by centrifugation. The melanin granules are then contacted with EDTA, the enzyme and DTT or DTE to release the analyte from the melanin granules, and the analyte analyzed by the methods described above.
The benefits to be obtained from use of the method according to the invention are many. The method provides a prompt and accurate diagnosis of prior exposure to a particular analyte. The subject hair and keratinized structure analysis method can provide a record of consumption, or non-consumption, over very long periods of time. Guess work regarding the true significance of one blood or urine analysis will be eliminated. Hair collection is less intrusive and less physically repulsive than blood or urine collection, and samples cannot be altered or substituted, nor can detection be evaded by short term abstention or xe2x80x9cflushingxe2x80x9d (excessive fluid intake) prior to a scheduled testing, e.g., pre-employment test or annual physical examination. Samples may be stored indefinitely without refrigeration.
The methods according to the invention, useful for the digestion of keratinized structures, e.g., hair, can also be used to ascertain the presence and structure of naturally occurring components of hair such as DNA.